Dissection of PIK3CA Aberration for Cervical Adenocarcinoma Outcomes.

Personalized treatment of genetically stratified subgroups has the potential to improve outcomes in many malignant tumors. This study distills clinically meaningful prognostic/predictive genomic marker for cervical adenocarcinoma using signature genomic aberrations and single-point nonsynonymous mutation-specific droplet digital PCR (ddPCR). Mutations in PIK3CA E542K, E545K, or H1047R were detected in 41.7% of tumors. PIK3CA mutation detected in the patient's circulating DNA collected before treatment or during follow-up was significantly associated with decreased progression-free survival or overall survival. PIK3CA mutation in the circulating DNA during follow-up after treatment predicted recurrence with 100% sensitivity and 64.29% specificity. It is the first indication of the predictive power of PIK3CA mutations in cervical adenocarcinoma. The work contributes to the development of liquid biopsies for follow up surveillance and a possibility of tailoring management of this particular women's cancer.

Molecular Characterization and Therapeutic Targeting of Colorectal Cancers Harboring Receptor Tyrosine Kinase Fusions.

PURPOSE: Receptor tyrosine kinase fusions in colorectal cancers are rare, but potentially therapeutically relevant. We describe clinical, molecular, and pathologic attributes of RTK fusion-associated colorectal cancer. EXPERIMENTAL DESIGN: We identified all cases with RTK fusions in patients with colorectal cancer seen at Dana-Farber Cancer Institute (Boston, MA) who underwent OncoPanel testing between 2013 and 2018. Clinical, histologic, and molecular features were extracted from the patient charts and molecular testing results. RESULTS: We identified 12 driver oncogenic fusions in various RTKs. These fusions occurred exclusively in BRAF and RAS wild-type tumors and were enriched in right-sided and mismatch repair-deficient (MMR-D) colorectal cancers. All of the MMR-D colorectal cancers with RTK fusions were found in tumors with acquired MMR-D due to MLH1 promoter hypermethylation and one was associated with a sessile serrated polyp. Molecular profiles of MMR-D colorectal cancer with RTK fusions largely resembled BRAF V600E-mutated MMR-D colorectal cancer, rather than those secondary to Lynch syndrome. We describe two patients with fusion-associated microsatellite stable (MSS) colorectal cancer who derived clinical benefit from therapeutic targeting of their translocation. The first harbored an ALK-CAD fusion and received sequential crizotinib and alectinib therapy for a total of 7.5 months until developing an ALK L1196Q gatekeeper mutation. The second patient, whose tumor contained an ROS1-GOPC fusion, continues to benefit from entrectinib after 9 months of therapy. CONCLUSIONS: RTK fusions in colorectal cancer are a rare, but important disease subgroup that occurs in RAS and BRAF wild-type tumors. Despite enrichment in acquired MMR-D tumors, RTK fusions also occur in MSS colorectal cancer and provide an important therapeutic target.

Insulin-Like Growth Factor-1 Receptor Expression and Disease Recurrence and Survival in Patients with Resected Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Insulin-like growth factor-1 receptor (IGF1R) signaling is important in pancreatic ductal adenocarcinoma (PDAC) biology, but little is known regarding IGF1R expression and patient characteristics and outcomes. METHODS: In 365 patients with resected PDAC, we evaluated IGF1R protein expression using IHC on whole-slide sections and IGF1R genomic status using next-generation sequencing. Associations of IGF1R expression, measured by H-scores incorporating staining intensity and proportion of positive tumor cells, with disease-free survival (DFS) and overall survival (OS) were evaluated in 317 and 321 patients, respectively, using Cox regression adjusting for known prognostic factors. RESULTS: Higher IGF1R expression in tumor cells was associated with worse DFS comparing highest versus lowest expression tertiles [median DFS, 10.8 vs. 16.1 months; adjusted hazard ratio (HR), 1.73; 95% confidence interval (CI), 1.24-2.44; P trend = 0.002] and worse OS (median OS, 17.4 vs. 25.8 months; HR, 1.39; 95% CI, 1.00-1.92; P trend = 0.046). The association between high IGF1R expression and reduced DFS was identified primarily among patients with a preoperative body mass index ≥25 kg/m2 (HR, 4.27; 95% CI, 2.03-8.96, comparing extreme tertiles; P interaction = 0.032). KRAS-mutant tumors had greater IGF1R expression, and IGF1R expression in tumor epithelium was inversely correlated with that in stromal cells. Mutations in IGF1R were infrequent, and no overt loss-of-function alterations were identified. Higher IGF1R expression was modestly associated with higher gene copy number (Pearson correlation coefficient = 0.26, P < 0.001). CONCLUSIONS: Higher IGF1R protein expression was associated with worse patient outcomes in resected PDAC. IMPACT: IGF1R expression in PDAC represents a potential biomarker to guide patient selection for more aggressive, multidrug regimens in the adjuvant setting.

ViroPanel: Hybrid Capture and Massively Parallel Sequencing for Simultaneous Detection and Profiling of Oncogenic Virus Infection and Tumor Genome.

Precision cancer medicine aims to classify tumors by site, histology, and molecular testing to determine an individualized profile of cancer alterations. Viruses are a major contributor to oncogenesis, causing 12% to 20% of all human cancers. Several viruses are causal of specific types of cancer, promoting dysregulation of signaling pathways and resulting in carcinogenesis. In addition, integration of viral DNA into the host (human) genome is a hallmark of some viral species. Tests for the presence of viral infection used in the clinical setting most often use quantitative PCR or immunohistochemical staining. Both approaches have limitations and need to be interpreted/scored appropriately. In some cases, results are not binary (virus present/absent), and it is unclear what to do with a weakly or partially positive result. In addition, viral testing of cancers is performed separately from tests to detect human genomic alterations and can thus be time-consuming and use limited valuable specimen. We present a hybrid-capture and massively parallel sequencing approach to detect viral infection that is integrated with targeted genomic analysis to provide a more complete tumor profile from a single sample.

Genomic analyses of flow-sorted Hodgkin Reed-Sternberg cells reveal complementary mechanisms of immune evasion.

Classical Hodgkin lymphoma (cHL) is composed of rare malignant Hodgkin Reed-Sternberg (HRS) cells within an extensive, but ineffective, inflammatory/immune cell infiltrate. HRS cells exhibit near-universal somatic copy gains of chromosome 9p/9p24.1, which increase expression of the programmed cell death protein 1 (PD-1) ligands. To define genetic mechanisms of response and resistance to PD-1 blockade and identify complementary treatment targets, we performed whole-exome sequencing of flow cytometry-sorted HRS cells from 23 excisional biopsies of newly diagnosed cHLs, including 8 Epstein-Barr virus-positive (EBV+) tumors. We identified significantly mutated cancer candidate genes (CCGs) as well as somatic copy number alterations and structural variations and characterized their contribution to disease-defining immune evasion mechanisms and nuclear factor κB (NF-κB), JAK/STAT, and PI3K signaling pathways. EBV- cHLs had a higher prevalence of genetic alterations in the NF-κB and major histocompatibility complex class I antigen presentation pathways. In this young cHL cohort (median age, 26 years), we identified a predominant mutational signature of spontaneous deamination of cytosine- phosphate-guanines ("Aging"), in addition to apolipoprotein B mRNA editing catalytic polypeptide-like, activation-induced cytidine deaminase, and microsatellite instability (MSI)-associated hypermutation. In particular, the mutational burden in EBV- cHLs was among the highest reported, similar to that of carcinogen-induced tumors. Together, the overall high mutational burden, MSI-associated hypermutation, and newly identified genetic alterations represent additional potential bases for the efficacy of PD-1 blockade in cHL. Of note, recurrent cHL alterations, including B2M, TNFAIP3, STAT6, GNA13, and XPO1 mutations and 2p/2p15, 6p21.32, 6q23.3, and 9p/9p24.1 copy number alterations, were also identified in >20% of primary mediastinal B-cell lymphomas, highlighting shared pathogenetic mechanisms in these diseases.

Genomic analyses of PMBL reveal new drivers and mechanisms of sensitivity to PD-1 blockade.

Primary mediastinal large B-cell lymphomas (PMBLs) are aggressive tumors that typically present as large mediastinal masses in young women. PMBLs share clinical, transcriptional, and molecular features with classical Hodgkin lymphoma (cHL), including constitutive activation of nuclear factor κB (NF-κB), JAK/STAT signaling, and programmed cell death protein 1 (PD-1)-mediated immune evasion. The demonstrated efficacy of PD-1 blockade in relapsed/refractory PMBLs led to recent approval by the US Food and Drug Administration and underscored the importance of characterizing targetable genetic vulnerabilities in this disease. Here, we report a comprehensive analysis of recurrent genetic alterations -somatic mutations, somatic copy number alterations, and structural variants-in a cohort of 37 newly diagnosed PMBLs. We identified a median of 9 genetic drivers per PMBL, including known and newly identified components of the JAK/STAT and NF-κB signaling pathways and frequent B2M alterations that limit major histocompatibility complex class I expression, as in cHL. PMBL also exhibited frequent, newly identified driver mutations in ZNF217 and an additional epigenetic modifier, EZH2. The majority of these alterations were clonal, which supports their role as early drivers. In PMBL, we identified several previously uncharacterized molecular features that may increase sensitivity to PD-1 blockade, including high tumor mutational burden, microsatellite instability, and an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) mutational signature. The shared genetic features between PMBL and cHL provide a framework for analyzing the mechanism of action of PD-1 blockade in these related lymphoid malignancies.

Recurrent genetic HLA loss in AML relapsed after matched unrelated allogeneic hematopoietic cell transplantation.

Immune evasion is a hallmark of cancer and a central mechanism underlying acquired resistance to immune therapy. In allogeneic hematopoietic cell transplantation (alloHCT), late relapses can arise after prolonged alloreactive T-cell control, but the molecular mechanisms of immune escape remain unclear. To identify mechanisms of immune evasion, we performed a genetic analysis of serial samples from 25 patients with myeloid malignancies who relapsed ≥1 year after alloHCT. Using targeted sequencing and microarray analysis to determine HLA allele-specific copy number, we identified copy-neutral loss of heterozygosity events and focal deletions spanning class 1 HLA genes in 2 of 12 recipients of matched unrelated-donor HCT and in 1 of 4 recipients of mismatched unrelated-donor HCT. Relapsed clones, although highly related to their antecedent pretransplantation malignancies, frequently acquired additional mutations in transcription factors and mitogenic signaling genes. Previously, the study of relapse after haploidentical HCT established the paradigm of immune evasion via loss of mismatched HLA. Here, in the context of matched unrelated-donor HCT, HLA loss provides genetic evidence that allogeneic immune recognition may be mediated by minor histocompatibility antigens and suggests opportunities for novel immunologic approaches for relapse prevention.

Germline cancer susceptibility gene variants, somatic second hits, and survival outcomes in patients with resected pancreatic cancer.

PURPOSE: Germline variants in double-strand DNA damage repair (dsDDR) genes (e.g., BRCA1/2) predispose to pancreatic adenocarcinoma (PDAC) and may predict sensitivity to platinum-based chemotherapy and poly(ADP) ribose polymerase (PARP) inhibitors. We sought to determine the prevalence and significance of germline cancer susceptibility gene variants in PDAC with paired somatic and survival analyses. METHODS: Using a customized next-generation sequencing panel, germline/somatic DNA was analyzed from 289 patients with resected PDAC ascertained without preselection for high-risk features (e.g., young age, personal/family history). All identified variants were assessed for pathogenicity. Outcomes were analyzed using multivariable-adjusted Cox proportional hazards regression. RESULTS: We found that 28/289 (9.7%; 95% confidence interval [CI] 6.5-13.7%) patients carried pathogenic/likely pathogenic germline variants, including 21 (7.3%) dsDDR gene variants (3 BRCA1, 4 BRCA2, 14 other dsDDR genes [ATM, BRIP1, CHEK2, NBN, PALB2, RAD50, RAD51C]), 3 Lynch syndrome, and 4 other genes (APC p.I1307K, CDKN2A, TP53). Somatic sequencing and immunohistochemistry identified second hits in the tumor in 12/27 (44.4%) patients with germline variants (1 failed sequencing). Compared with noncarriers, patients with germline dsDDR gene variants had superior overall survival (hazard ratio [HR] 0.54; 95% CI 0.30-0.99; P = 0.05). CONCLUSION: Nearly 10% of PDAC patients harbor germline variants, although the majority lack somatic second hits, the therapeutic significance of which warrants further study.

Duodenal-type and nodal follicular lymphomas differ by their immune microenvironment rather than their mutation profiles.

Duodenal-type follicular lymphoma (DTFL) is a rare and highly indolent follicular lymphoma (FL) variant. It is morphologically and immunophenotypically indistinguishable from typical FL, characterized by restricted involvement of intestinal mucosa, and lacks extraintestinal manifestations. The molecular determinants of this distinct clinical behavior are largely unknown. Thirty-eight diagnostic biopsies from patients with DTFL were evaluated. The 10-year overall survival rate was 100% in clinically evaluable patients (n = 19). We compared the targeted mutation profile of DTFL (n = 31), limited-stage typical FL (LSTFL; n = 17), and advanced-stage typical FL (ASTFL; n = 241). The mutation frequencies of recurrently mutated genes, including CREBBP, TNFRSF14/HVEM, and EZH2 were not significantly different. However, KMT2D was less commonly mutated in DTFL (52%) and LSTFL (24%) as compared with ASTFL (79%). In ASTFL, 41% of KMT2D-mutated cases harbored multiple mutations in KMT2D, as compared with only 12% in LSTFL (P = .019) and 0% in DTFL (P < .0001). Whole exome and targeted sequencing of DTFL revealed high mutation frequencies of EEF1A1 (35%) and HVCN1 (22%). We compared the immune microenvironment gene expression signatures of DTFL (n = 8) and LSTFL (n = 7). DTFL clearly separated from LSTFL by unsupervised, hierarchical clustering of 147 chemokines and cytokines and was enriched for a chronic inflammation signature. In conclusion, the mutational landscape of DTFL is highly related to typical FL. The lower frequency of multiple mutations in KMT2D in DTFL and LSTFL indicates an increasing selection pressure for complete KMT2D loss in ASTFL pathogenesis. The highly dissimilar immune microenvironment of DTFL suggests a central role in the biology of this disease.

Author Correction: Molecular subtypes of diffuse large B cell lymphoma are associated with distinct pathogenic mechanisms and outcomes.

In the version of this article originally published, an asterisk was omitted from Fig. 1a. The asterisk has been added to the figure. Additionally, a "NOTCH2" label was erroneously included in Fig. 4a. The label has been removed. The errors have been corrected in the PDF and HTML versions of this article.